Development of Zika viral pseudoinfectious virus as zika vaccine candidate Abstract Zika virus(ZIKV) is a mosquito-borne flavivirus that was first identified in Uganda in 1947. It then spread to Asia and Pacific. Recently, the explosive outbreaks in Americas and its implication in Guillan Barr (GBS) syndrome and the birth of microcephalic infants following maternal infection lead to WHO declared that ZIKV infection is ?a Public Health Emergency of International Concern?. Currently, there is no specific treatment. Thus, developing a safe and effective vaccine for ZIKV infection is a high priority for public health. Here, we propose to develop a novel ZIKV vaccine candidate based on ZIKV pseudoinfectious virus (PIV) produced by incorporation of ZIKV subgenomic replicon into viral particle in stable packaging cells. The approach is based on our extensive experience of dengue virus (DENV) PIV investigation. In DENV PIV production system, the subgenomic replicon was constructed by deleting the capsid protein (C) gene from the DENV genome and optimizing the signal peptide sequence of pre- membrane protein (prM) to facilitate the formation of viral particles. Packaging cells were developed for inducible expression of a bi-protein Cpr, where the protein pr is the ?pr? segment of viral protein prM that holds the protein C on the endoplasmic reticulum (ER). When the replicon was introduced into the packaging cells, protein C was released from the bi-protein Cpr by a replicon-encoded viral protease. Coordinate expression of viral structural proteins by the replicon and packaging cells led to the incorporation of the replicon into viral particle to produce DENV PIVs. Toward to develop a ZIKV PIV, we have constructed a full-length cDNA clone of ZIKV by chemical synthesis. Based upon these highly promising results, the Specific Aims of this Phase I STTR proposal are: 1) construction of subgenome for ZIKV 2) development of stable packaging cell lines based on FDA approved Vero cells, 3) rapid and efficient harvesting of ZIKV PIV from infected packaging lines, and 4) preliminary analysis of the safety and immunogenicity of the proposed vaccine in mice. Successful completion of this program will enable a Phase II research including development of a cost-effective GMP process for PIVs production at industrial scale and a pre-clinical animal study of immunity to ZIKV infection.